Antiradical Capacity and Induction of Apoptosis on HeLa Cells by ...

Plant Foods Hum Nutr (2008) 63:35–40 DOI 10.1007/s11130-007-0066-4

ORIGINAL PAPER

Antiradical Capacity and Induction of Apoptosis on HeLa Cells by a Phaseolus vulgaris Extract Xochitl Aparicio-Fernández & Rosalia Reynoso-Camacho & Eduardo Castaño-Tostado & Teresa García-Gasca & Elvira González de Mejía & S. Horacio Guzmán-Maldonado & Guillermo Elizondo & Gad Gabra Yousef & Mary Ann Lila & Guadalupe Loarca-Pina

Published online: 19 December 2007 # Springer Science + Business Media, LLC 2007

Abstract Jamapa bean is a black Phaseolus vulgaris variety rich in condensed tannins, anthocyanins and flavonols with interesting biological activities. The objective of this work was to evaluate the antiradical capacity (ARC) of a Jamapa bean methanolic extract (BME) and some of the proanthocyanidin-rich fractions derived from it, using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay. The effect of the BME on some proteins involved in apoptosis on HeLa cells was also evaluated. A strong correlation between proanthocyanidin concentration in BME and antiradical capacity was found, suggesting that these compounds contribute significantly to antiradical activity. BME was a better radical scavenger than butylated hydroxytoluene (45.6 and 33.9% ARC at 400 µM, respec-

X. Aparicio-Fernández : R. Reynoso-Camacho : E. Castaño-Tostado : G. Loarca-Pina (*) Programa de Posgrado en Alimentos del Centro de la Republica (PROPAC), Research and Graduate Studies in Food Science, School of Chemistry, Universidad Autonoma de Queretaro, Queretaro, Qro. 76010, Mexico e-mail: [email protected] T. García-Gasca Nutrition School, Natural Sciences Faculty, Universidad Autonoma de Queretaro, Queretaro, Qro. 76010, Mexico E. González de Mejía Department of Food Science and Human Nutrition, University of Illinois at Urbana-Champaign, 228 ERML, 1201 W Gregory Drive, Urbana, IL 61801, USA

tively). Two proanthocyanidin-rich fractions obtained after a preliminary separation of the BME using Toyopearl (TP4 and TP6) exhibited a higher antiradical activity than the parent extract. The treatment of HeLa cells with 35 µg BME/ml/24 h increased the expression of Bax and Caspase3, pro-apoptotic proteins (6.13 and 1.2 times for Caspase-3 and Bax, respectively). The mechanism of action of some proteins involved in apoptosis was also evaluated, and the results suggest that black Jamapa bean could be an important source of polyphenolic compounds with potential biological use as antioxidant and anticancer agents. Keywords Antiradical capacity . Apoptosis . HeLa cells . Phaseolus vulgaris L. . Phenolic compounds

S. H. Guzmán-Maldonado Biotechnology, Legume and Plant Nutrition Laboratories, Experimental Station El Bajío, National Research Institute for Forestry, Agriculture and Livestock (INIFAP), Celaya, Mexico G. Elizondo Toxicology Section, CINVESTAV-IPN, Ave. IPN 2508, México, D.F. 06380, Mexico G. G. Yousef : M. A. Lila Department of Natural Resources and Environmental Sciences, University of Illinois at Urbana-Champaign, 1201 South Dorner Drive, Urbana, IL 61801, USA

36

Abbreviations ARC Antiradical capacity BHT Butylated hydroxytoluene BME Bean methanolic extract DPPH 1,1-diphenyl-2-picrylhydrazyl

Introduction Epidemiological evidence consistently shows that the ingestion of a diet rich in plant foods (including fruits, vegetables, legumes and cereals) is strongly associated with reduction in the risk of developing chronic diseases, such as cancer, diabetes, obesity and cardiovascular diseases [1–3]. The information supports that around two-thirds of cancerrelated deaths could be prevented through lifestyle modification, particularly through dietary means [4]. The protecting effect of plant foods has been related to the presence of phytochemicals, bioactive non-nutrient plant compounds [5]. Phytochemicals can be classified as carotenoids, phenolics, alkaloids, nitrogen-containing compounds, and organosulfur compounds, which commonly can have complementary and overlapping mechanisms of action including radical scavenging, antimutagenic activities, and induction of apoptosis in cancer cell lines, among others [6]. The most studied groups of phytochemicals are phenolics and carotenoids. Polyphenolics, including flavonoids, are present in a wide range of plants such as legumes. Flavonoids are protective or defensive compounds for the host plant, protecting against insect or pathogen attack, or harmful UV radiation, providing antioxidant protection, or guiding pollinators [7, 8]. The daily intake of flavonoids for humans can be as high as 1–2 g [8]. Common bean (Phaseolus vulgaris L.) is an important protein, carbohydrate and vitamin source in Mexico and some other Latin American countries [9]. In addition, the presence of bioactive phytochemicals in beans, such as trypsin inhibitors, lectins and phenolic compounds, has been recently described [10, 11]. The presence of different types of polyphenolics in common beans has been widely reported [10, 12]. The biological activity of flavonoid rich extracts from common beans includes antimutagenicity against different mutagens, such as benzo[a]pyrene, 1nitropyrene and aflatoxin B1 (AFB1) [13, 14], antioxidant activity [15, 16], and antiproliferative effect on cancer cell lines [17–19]. In a previous work [12], the methanolic extract obtained from the testa of black Jamapa common beans (BME) was fractionated to further characterize its composition; vacuum liquid chromatography was employed using Toyopearl chromatography resin. Several flavonoids were characterized from BME including anthocyanins, proanthocyanidins and flavonols. Toyopearl (TP) fractions were tested for

Plant Foods Hum Nutr (2008) 63:35–40

biological activity on HeLa cancer cells. The fractions with the highest proportion of proanthocyanidin TP4 (54.6%) and TP6 (58.9%) presented inhibitory activity on cancer cells [18]. The whole BME also presented important antimutagenic activity against AFB1 [20], inhibitory activity against HeLa cancer cells and to a lesser extent against HaCaT premalignant cells [18]. Nevertheless, potential mechanisms by which these flavonoids accomplish apoptosis have not been elucidated. It has been reported that vegetable polyphenolics can induce growth arrest and apoptosis through regulation of multiple signaling pathways [21], in which reactive oxygen species seem to be indispensable [22]. Their effect could be at different cancer stages including inhibiting oxygen radical-forming enzymes [23] and antiradical/antioxidant activity could be partially related to apoptosis induction. The aim of this work was to evaluate the antiradical capacity of Jamapa BME and to examine its effect on some proteins involved in apoptosis on HeLa cells. The antiradical capacity of the proanthocyanidin-rich fractions with high inhibitory activity on cancer cells was also evaluated.

Materials and Methods Plant Material Seeds from Jamapa, a black common bean variety (P. vulgaris L.) were grown in 2003 at “El Bajío” Experimental Station of the National Research Institute for Forestry Agriculture and Livestock (INIFAP), Mexico. Dry seeds were stored at −20 °C until manual separation of the seed coats. The seed coats were lyophilized and stored in the dark at −20 °C until extraction and further analysis. Chemicals Butylated hydroxytoluene (BHT), 1,1-diphenyl2-picrylhydrazyl (DPPH), (+)-catechin, 6-hydroxy-2,5,7,8tetramethylchroman-2-carboxylic acid (trolox), penicillin, streptomycin and comptothecin were from Sigma Chemical Co (St. Louis, MO). Dulbecco’s Modified Eagle’s Medium (DMEM), bovine serum albumin (BSA) and heat inactivated fetal bovine serum (FBS) were obtained from Gibco BRL (Grand Island, NY). Electrophoresis chemicals, acrylamide, bis-acrylamide, TEMED, ammonium persulfate, tris, Coomasie blue G-250 and bromophenol blue were from BioRad (Hercules, CA). Primary and secondary antibodies, anti-Bax, anti-Caspase-3 and anti β-Actin were from Santa Cruz Laboratories (Santa Cruz, CA) and Zymed. Extraction of Polyphenolics from Jamapa Bean Seed Coats Extraction of polyphenolic compounds from Jamapa bean testa was done following the protocol previously described by Cardador-Martínez et al. [14]. Condensed tannins were quantified according to Deshpande and


37

Table 1 Antiradical capacity of methanolic extract from common beans (P. vulgaris cv Jamapa) (BME), (+)-catechin and butylated hydroxytoluene (BHT) Concentration µM1

50 100 200 400 600 800 1000

Antiradical capacity (ARC %) BME

(+)-catechin

BHT

ND2 10.0±0.17i,j 22.5±0.37f,g,h,i 45.6±0.70c,d,e 61.0±0.90b,c 76.7±1.00a,b 88.3±0.90a

12.8±0.40h,i,j 31.0±0.82e,f,g 50.7±1.29c 77.0±1.52a,b 89.8±0.83a 91.7±0.34a 92.3±0.12a

0.8±0.17j 7.6±0.34i,j 16.5±0.70g,h,i,j 33.9±1.41d,e,f 49.2±1.95c,d 57.9±2.28c ND2

1 Molar concentration of BME was calculated as (+)-catechin equivalents. Each value represents the mean of three independent experiments ± variability coefficient. Means in a column followed by the same letter are not significantly different (Tukey, α=0.05) 2 Not determined

Cheryan [24, 25] and reported as mg of (+)-catechin equivalent per gram of lyophilized extract. Fractionation of BME BME fractionation was done according to Aparicio-Fernandez et al. [12]. TP4 and TP6 fractions were chosen for antiradical analysis based on their chemical composition (mainly proanthocyanidins) [12] and their biological activity on cancer cells [18]. DPPH Method Antioxidant capacity by the DPPH method [26] was adapted for use with microplates as described previously [27]. Sample concentrations tested were 0– 1,000 µM catechin equivalents based on phenolic content. Antiradical capacity (ARC) was calculated according to the equation of Burda and Oleszek [28]: ARC ¼ 100 ð1 absorbance sample=absorbance of controlÞ. Cell Culture and Treatment HeLa, human cervix adenocarcinoma cells (ATCC, CCL-2), were cultured as described by Aparicio-Fernández et al. [18]. The treatment was performed for 24 h with 35 µg of dry BME per ml directly dissolved in DMEM containing 1% BSA. Controls consisted of untreated cells. Camptothecin (5 µM) an anticancer agent were used as positive control Effect of Methanolic Extract Treatment on Caspase-3 and Bax Proteins Expression HeLa cells were plated in 60 mm Petri dishes at 2×105 cells/ml in 10% FBS DMEM. Cells were incubated and medium was refreshed every other day until preconfluence, when they were treated as previously described. After treatment, adherent cells were washed with PBS and resuspended in 200 µl lysis buffer (70 mM sucrose, 1 mM EDTA, 10 mM HEPES, 1% nonidet P40, 1 mM sodium ortovanadate, 1 mM fenilmetilsulfonil fluoride, 10 µg/ml leupeptine). Cells were lysed by passing through a syringe needle. Samples were allowed to stand

for 30 min on ice, and then were centrifuged at 13,000×g, for 10 min, at 4 °C. Total cell extract was subjected to protein content analysis by the Lowry method [29]. Samples were electrophoresed in a 10% SDS-PAGE gel (120 V, 180 min), and transferred onto a polyvinyldifluoride membrane (15 V, 10 min). The membrane was saturated over a period of 3 h at room temperature with 5% blocking solution and incubated 3 h with anti-Bax and anti-Caspase3 monoclonal antibodies (diluted 1:500 and 1:1,000, respectively). After several washes with phosphate saline solution, the membranes were incubated for 1 h in the presence of rabbit anti-mouse IgG antibody conjugated to horseradish peroxidase (diluted 1:5,000 and 1:2,000, Bax and Caspase 3, respectively). Visualization of immunoreactive bands was accomplished using a chemiluminiscence kit (MB Chemiluminiscence Blotting Substrate (POD), Roche Diagnosis Co, IN) and Kodak photographic film (BioMax Light Film, Eastman Kodak Co, NY). The protein expression quantification was done using image-processing software (IPLab, Scanalytics, Billerica, MA), and is reported as a percentage of the control.

Table 2 Antiradical capacity of methanolic extract from common beans (P. vulgaris cv Jamapa) (BME), Toyopearl fraction 4 (TP4) and Toyopearl fraction 6 (TP6) Sample1

Antiradical activity (ARC %)

BME TP4 TP6

61.4±0.8b 79.8±0.94a 85.4±0.95a

Each value represents the mean of three independent experiments ± standard error. Means in a column followed by the same letter are not significantly different (Tukey, α=0.05) 1 All samples were tested at 208 µg/ml concentration

38


Table 3 Correlation coefficients for phenolics in methanolic extract from common beans (P. vulgaris cv Jamapa) (BME), butylated hydroxytoluene (BHT) and (+)-catechin with antiradical capacity (ARC) ARC BMEa BHT (+)-catechin a

0.99 0.99 0.93

Expressed as µM equivalent to (+)-catechin

Statistical Analysis Results are expressed as mean ± standard error. Statistical significance was determined using Tukey’s procedure (α=0.05) [30].

Results and Discussion Concentration of proanthocyanidins in BME was 586± 93.7 mg of (+)-catechin equivalent per one g of lyophilized extract, as reported in our previous work [20]. The free radical scavenging activity of BME and standards (BHT and (+)-catechin) was tested by measuring their ability to quench the stable DPPH radical. This assay provides stoichiometric information with respect to the number of electrons taken up by the tested compounds in the presence of the stable radical. Table 1 shows the ARC, expressed as a percentage, for BME, (+)-catechin and BHT. The ARC of BME, at all concentrations tested, was higher than the ARC of BHT at the same concentrations, but lower than the ARC of (+)-catechin. This suggests a better capacity of proanthocyanidins present in BME to quench free radicals compared to BHT, and lower antiradical capacity of BME compared to (+)-catechin. This antioxidant behavior of BME compared to standards is similar to the data reported Fig. 1 Effect of 24 h treatment with methanolic extract from common beans (P. vulgaris cv Jamapa) (BME) on Caspase-3 (a) and Bax (b) proteins expression in HeLa cells. Control: protein expression in cells without any treatment; BME: protein expression induced by treatment with 35 µg BME/ml; Camp: protein expression induced by anticancer drug camptothecin (5 µM). β-Actin expression was monitored as control

by Cardador-Martínez et al. [27], which demonstrates the reproducibility of the DPPH assay. ARC of Jamapa BME at 100 µM (10±0.17%) was within the range reported by Oomah et al. [15] for six Canadian bean varieties (ARC= 4.61 to 17.41%). The theoretically calculated value for ARC=50% (EC50) was 508.7 µM, which corresponded to 208 µg BME per ml. This concentration was chosen to test the antiradical capacity of TP4 and TP6 fractions obtained during characterization and determined to be composed of primarily proanthocyanidins (54.6 and 58.9% of proanthocyanidin content for TP4 and TP6, respectively) [12]. In our previous work, these fractions demonstrated inhibitory potency on HeLa cancer cell proliferation [18]. Table 2 shows that ARC of TP4 and TP6, fractions was significantly higher than the ARC of BME. The differences in ARC value of samples could be due to a concentration effect of proanthocyanidins, as well as the presence of different types of proanthocyanidins among analyzed samples (TP4, TP6 and BME). It has been reported that the antiradical capacity is strongly affected by the sample composition [16, 31]. The concentration of the BME, expressed as µM equivalent to (+)-catechin (100–1,000 µM), showed a significant correlation with antiradical capacity (ARC) (R= 0.99) suggesting that the antiradical activity of the BME is mainly provided by the polyphenolic compounds (Table 3). The results are in accordance with the investigation reported by Oomah et al. [15], who found antiradical activity in six different bean varieties grown in Canada, which was strongly correlated to flavonoid content. In other legumes such as peas, antioxidant activity has been related to the presence of condensed tannins; and it was noted that legumes with colored seed coats show higher proanthocyanidin content and higher antioxidant activity evaluated in different systems [16, 32]. Standards, BHT (50–800 µM) and (+)-catechin (50–600 µM), had high correlations between compound


concentration and antiradical capacity (R=0.99 and 0.93, for BHT and (+)-catechin, respectively), which is consistent with previous reports [26, 28] about antioxidant capacity of pure phenolic compounds. In a previous work [18], it was reported that BME inhibited the proliferation of HeLa cells (IC50 =41.3 µg BME/ml at 24 h treatment), and cell cytometry analysis showed that the treatment of HeLa cells with 35 µg BME/ ml over an interval of 24 h, increased DNA molecule breakage in cells from 2.9 to 22.1%. These results suggested that the BME (35 µg/ml) caused apoptosis in the cells, therefore, the effect of BME treatment on the expression of Bax and Caspase-3 proteins in HeLa cells was evaluated and compared to treatment with camptothecin, an anticancer drug used as control. To ensure equal loading of proteins in all the samples, a β-actin control was used. The western blot analysis revealed that treatment of HeLa cells with BME produced an alteration in the expression of Bax and Caspase-3 proteins (Fig. 1). Expression levels of Caspase-3 protein increased around 6.13 times respect to control cells expression after treatment with BME (Fig. 1a). In the same way camptothecin treatment raised Caspase-3 expression (4.9 times respect to control cells expression). The expression of Bax was positively affected too, although to a lesser extent (Fig. 1b). Bax expression increased to 1.2 times in HeLa cells after treatment with BME, meanwhile the treatment with camptothecin raised the expression of Bax to 2.4 times respect to control cells. β-Actin protein remained unaffected in the presence of BME and camptothecin (Fig. 1). Polyphenolic-rich extracts from different vegetable sources have demonstrated induction of apoptosis through caspase3 and Bax pathways in cancer cell lines [33, 34]. Bax is a pro-apoptotic member of the Bcl-2 protein family that resides in the outer mitochondrial membrane. In many types of apoptosis, Bax undergoes a change in conformation, forming a high weight molecular complex in outer mitochondrial membrane [35]. This event always precedes the release of mitochondrial cytochrome c, which, in the cytosol, activates caspases through binding to Apaf-1 [36]. The released cytochrome c is associated with ion channel (MAC) activity in the outer membrane of mitochondria and with the onset of apoptosis [37]. Caspase-3 is required for DNA fragmentation and some of the typical morphological changes of cells undergoing apoptosis [38]. These results clarify the apoptosis induction of BME on HeLa cells. The BME, which is a mixture of anthocyanins, proanthocyanins and flavonols, exhibited antiradical activity to a higher extent than BHT, a commercial and synthetic antioxidant used in food industry. Fractionation of BME using liquid chromatography yielded fractions, enriched in proanthocyanins, with an increased antiradical activity (TP4 and TP6). The same behavior was observed on the

39

inhibitory activity of fractions on HeLa cells proliferation [18]. On the other hand, it has been described that the prooxidant action of plant-derived phenolics, rather than their antioxidant action, may be an important mechanism for their anticancer and apoptosis-inducing properties, as reactive oxygen species can mediate apoptotic DNA fragmentation and their pro-oxidant phenoxyl radicals can cause mitochondrial toxicity by collapsing the mitochondrial membrane potential [23]. In this research, the induction of apoptosis in HeLa cells by BME through mitochondrial pathway was evidenced. More research is needed in order to completely elucidate the BME mechanism of action on HeLa cells inhibition. The results suggest that the black Jamapa bean could be an important source of polyphenolic compounds with potential use as pro-oxidants/ antioxidants and anticancer activity. This study indicated that polyphenol-rich fractions from Jamapa beans can be used as nutraceutical agents for food and/or pharmaceutical industry. Acknowledgements The authors wish to thank Consejo Nacional de Ciencia y Tecnología (CONACYT) for supporting the work under grant 31623-B, and “El Bajío” Experimental Station of the National Research Institute for Forestry Agriculture and Livestock (INIFAP) for black Jamapa beans donation.

References 1. Chen Ch, Kong AT (2005) Dietary cancer-chemopreventive compounds: from signaling and gene expression to pharmacological effects. Trends Pharmacol Sci 26(6):318–326 2. Adams SM, Standridge JB (2006) What should we eat? Evidence from observational studies. South Med J 99(7):744–748 3. Fernández E, Gallus S, La Vecchia C (2006) Nutrition and cancer risk: an overview. J Br Menopause Soc 12(4):139–142 4. Barnard RJ (2004) Prevention of cancer through lifestyle changes. Evid Based Complement Alternat Med 1(3):233–239 5. Liu RH (2003) Health benefits of fruits and vegetables are from additive and synergistic combination of phytochemicals. Am J Clin Nutr 78:517S–520S 6. Liu RH (2004) Potential synergy of phytochemicals in cancer prevention: mechanism of action. J Nutr 134:3479–3485 7. Wildman REC (2001) Classifying nutraceuticals. In: Wildman REC (ed) Handbook of nutraceuticals and functional foods. CRC Press, Boca Raton, FL (pp 13–30) 8. Havsteen BH (2002) The biochemistry and medical significance of the flavonoids. Pharmacol Ther 96(2–3):67–202 9. Serrano J, Goni I (2004) Role of black bean (Phaseolus vulgaris) on the nutritional status of Guatemalan population. Arch Latinoam Nutr 54(1):36–44 10. Beninger CW, Hosfield GL (2003) Antioxidant activity of extracts, condensed tannin fractions, and pure flavonoids from Phaseolus vulgaris L. seed coat color genotypes. J Agric Food Chem 51:7879–7883 11. De Mejia EG, Del Carmen Valadez-Vega M, Reynoso-Camacho R, Loarca-Pina G (2005) Tannins, trypsin inhibitors and lectin cytotoxicity in tepari (Phaseolus acutifolius) and common (Phaseolus vulgaris) beans. Plant Foods Hum Nutr 60(3):137–145 12. Aparicio-Fernandez X, Yousef GG, Loarca-Pina G, de Mejia E, Lila MA (2005a) Characterization of polyphenolics in the seed

40

13.

14.

15.

16.

17.

18.

19.

20.

21.

22. 23.

24.

Plant Foods Hum Nutr (2008) 63:35–40 coat of black Jamapa bean (Phaseolus vulgaris L.). J Agric Food Chem 53:4615–4622 González de Mejía E, Castaño-Tostado E, Loarca-Piña G (1999) Antimutagenic effects of natural phenolic compounds in beans. Mut Res Genet Toxicol Environ Mutagen 441(1):1–9 Cardador-Martínez A, Castaño-Tostado E, Loarca-Piña G (2002a) Antimutagenic activity of natural phenolic compounds present in common bean (Phaseolus vulgaris L.) against Aflatoxin B1. Food Addit Contam 19:62–69 Oomah BD, Cardador-Martínez A, Loarca-Pina G (2005) Phenolics and antioxidative activities in common beans (Phaseolus vulgaris L.). J Sci Food Agric 85:935–942 Galvez-Ranilla L, Genovese MI, Lajolo FM (2007) Polyphenols and antioxidant capacity of seed coat and cotyledon from Brazilian and Peruvian bean cultivars (Phaseolus vulgaris L.). J Agric Food Chem 55:90–98 Bawadi HA, Bansode RR, Trappey IIA, Truax RE, Losso JN (2005) Inhibition of Caco-2 colon, MCF-7 and Hs578T breast, and DU 145 prostatic cancer cell proliferation by water-soluble black bean condensed tannins. Cancer Lett 218:153–162 Aparicio-Fernandez X, García-Gasca T, Yousef GG, Lila MA, de Mejia E, Loarca-Pina G (2006) Chemopreventive activity of polyphenolics from black Jamapa bean (Phaseolus vulgaris L.) on HeLa and HaCaT cells. J Agric Food Chem 54:2116–2122 Dong M, He X, Liu RH (2007) Phytochemicals of black bean seed coats: isolation, structure elucidation, and their antiproliferative and antioxidative activities. J Agric Food Chem 55:6044– 6051 Aparicio-Fernandez X, Manzo-Bonilla L, Loarca-Piña G (2005b) Comparison of antimutagenic activity of phenolic compounds in newly harvested and stored common beans Phaseolus vulgaris against aflatoxin B1. J Food Sci 70:S73–S78 Shankar S, Ganapathy S, Srivastava RK (2007) Green tea polyphenols: biology and therapeutic implications in cancer. Front Biosci 12:4884–4899 Fang J, Nakamura H, Iyer AK (2007) Tumor-targeted induction of oxystress for cancer therapy. J Drug Target 15:475–486 Galati G, O’Brien PJ (2004) Potential toxicity of flavonoids and other dietary phenolics: significance for their chemopreventive and anticancer properties. Free Radic Biol Med 37(3):287–303 Deshpande SS, Cheryan M (1985) Evaluation of vanillin assay for tannin analysis of dry beans. J Food Sci 50:905–910

25. Deshpande SS, Cheryan M (1987) Determination of phenolic compounds of dry beans using vanillin, redox and precipitation assays. J Food Sci 52(2):332–334 26. Fukumoto LR, Mazza G (2000) Assessing antioxidant and prooxidant activities of phenolic compounds. J Agric Food Chem 48:3597–3604 27. Cardador-Martínez A, Loarca-Piña G, Oomah BD (2002b) Antioxidant activity in common beans (Phaseolus vulgaris L.). J Agric Food Chem 50:6975–6980 28. Burda S, Oleszek W (2001) Antioxidant and antiradical activities of flavonoids. J Agric Food Chem 49:2774–2779 29. Lowry OH, Rosenbrough JN, Fan A, Randall RJ (1951) Protein measurement with folin phenol reagent. J Biol Chem 193:265–275 30. SAS Institute (1999) SAS user’s guide: Statistics, 8th edn. SAS Institute, Cary NC 31. Heimler D, Vognolini P, Dini MG, Romani A (2005) Rapid tests to assess the antioxidant activity of Phaseolus vulgaris L. dry beans. J Agric Food Chem 53:3053–3056 32. Troszyńska A, Ciska E (2002) Phenolic compounds of seed coats of white and coloured varieties of pea (Pisum sativum L.) and their total antioxidant activity. Czech J Food Sci 20:15–22 33. Kundu T, Dey D, Roy M, Siddiqi M, Bhattacharya RK (2005) Induction of apoptosis in human leukemia cells by black tea and its polyphenol theaflavin. Cancer Lett 230:111–121 34. Roy AM, Baliga MS, Elmets CA, Katiyar SK (2005) Grape seed proanthocyanidins induce apoptosis through p53, Bax, and Caspase 3 pathways. Neoplasia 7(1):24–36 35. Antonsson B, Montessuit S, Sanchez B, Martinou JC (2001) Bax is present as a high molecular weight oligomer/complex in the mitochondrial membrane of apoptotic cells. J Biol Chem 276 (15):11615–11623 36. Eskes R, Desagher S, Antonsson B, Martinou JC (2000) Bid induces the oligomerization and insertion of Bax into the outer mitochondrial membrane. Mol Cell Biol 20(3):929–935 37. Pavlov EV, Priault M, Pietkiewicz D, Cheng EHY, Antonsson B, Manon S, Korsmeyer SJ, Mannella CA, Kinnally KW (2001) A novel, high conductance channel of mitochondria linked to apoptosis in mammalian cells and Bax expression in yeast. J Cell Biol 155(5):725–731 38. Jänicke RU, Sprengart ML, Wati MR, Porter AG (1998) Caspase-3 is required for DNA fragmentation and morphological changes associated with apoptosis. J Biol Chem 273(16):9357–9360